title: [Congenital afibrinogenemia caused by a novel insertion mutation in the FGB gene].
abstract: OBJECTIVE
  To investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia.
  
  
  METHODS
  The plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay, respectively. Genomic DNA was isolated from peripheral blood of the proband and his related family members. All exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Thrombin fibrin aggregation curve were detected in the plasma of the patient. Wild-type and mutation type fibrinogen vectors were constructed, and then transfected into COS-7 cells. The wild-type and mutant proteins from the culture media and cell lysates were tested by Western blot and ELISA.
  
  
  RESULTS
  APTT, PT, TT were significantly longer in the proband. Plasma fibrinogen activity and antigen of the patient could not be detected using the Clauss method and immuno-nephelometry, respectively. Gene analysis revealed that a novel homozygous GTTT insertion between nucleotides 2833 and 2834 in FGB exon 2 in the proband. The proband's father, mother, brother and son were heterozygous. The polymerization curves of the patient did not show a lag phase or final turbidity, compared with the normal controls. Western blot analysis showed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen in patient's plasma under non-reducing conditions. It also could not detect the truncated Bβ chain under reducing conditions. Abnormal fibrinogen molecule (molecule weight>340 000) were found in transfected COS-7 cells by Western blot, which indicated that the mutation caused the abnormal intracellular fibrinogen molecule assembly. The fibrinogen band was absent in culture media transfected by the mutation. Fibrinogen levels of mutant fibrinogen were no significant different from those of wild-type fibrinogen in cell lysates by ELISA analysis [(2.47 ± 0.30) μg/ml vs (2.65±0.60) μg/ml, P=0.0889]; However, the levels of the mutant fibrinogen were statistically significant lower than those of wild type fibrinogen in culture media [(0.01 ± 0.01) μg/ml vs (3.80±0.80) μg/ml, P=0.0001].
  
  
  CONCLUSIONS
  Congenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB. This novel mutation impaired fibrinogen assembly and secretion.
authors.affiliation: Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, Suzhou 215006, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Xiao-juan
authors.initials: XJ
authors.lastname: Zhao
authors.forename: Zhao-yue
authors.initials: ZY
authors.lastname: Wang
authors.forename: Zi-qiang
authors.initials: ZQ
authors.lastname: Yu
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Cao
authors.forename: Zhen-ni
authors.initials: ZN
authors.lastname: Ma
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Xia
authors.initials: X
authors.lastname: Bai
authors.forename: Chang-geng
authors.initials: CG
authors.lastname: Ruan
mesh_terms: D000347:Afibrinogenemia / Q000151:congenital* / Q000209:etiology / Q000235:genetics; D005340:Fibrinogen / Q000235:genetics*; D016368:Frameshift Mutation; D006801:Humans; D008297:Male; D016254:Mutagenesis, Insertional; D010375:Pedigree; D055815:Young Adult
chemical_list: D005340:Fibrinogen